Western blot

2 × 10⁶ cells/sample were resuspended in 100 µL sampling buffer (65 mM Tris-HCl [pH 6.8], 2% SDS, 1 mM Na₃VO₄, 26% glycerol, 1% β-mercaptoethanol and 0.01% bromphenol blue), lysed by sonification using Vibra Cell sonicator (Sonics & Materials, Newtown, CT, USA) and heated for 5 min at 95 °C. The sample volume used was defined by the protein concentration, which was determined by Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A color prestained protein standard (New England BioLabs, Frankfurt a.M., Germany) and the samples were run on a 10% polyacrylamide gel at 170 V and blotted onto nitrocellulose membranes at 100 V. Loading of the gel and protein transfer was confirmed by Ponceau S staining (AppliChem). Membranes were blocked for 1 h in TBS-T (20 mM Tris [pH 7.6],137 mM NaCl and 0.1% Tween-20), containing 5% fat-free dry milk. Subsequently, membranes were incubated overnight at 4 °C in primary antibodies which were diluted 1:1000 in TBS-T and 5% bovine serum albumin (p38 MAPK (Cat. 9212 S), phospho-p38 MAPK (Thr180/Tyr182) (Cat. 9215 S), T-bet (Cat. 13232), GATA-3 (Cat. 5852), FoxP3 (Cat. 12632), β-actin (Cat. 4967 S) (all from Cell Signaling Technology, Danvers, MA, USA), RORγ(t) (Cat. PA5-86733, Invitrogen by Thermo Fisher Scientific)). The secondary antibody HRP-linked anti-rabbit IgG (Cat. 7074, Cell Signaling Technology) was diluted 1:2000 in TBS-T and 5% fat-free dry milk and membranes were incubated at room temperature for 3 h. Band density was determined using Westar Antares (Cyanagen, Bologna, Italy) and LAS-3000 (Fujifilm) and was analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA). Original western blots are available as supplementary information.